Urokinase inhibitors and uses thereof

ABSTRACT

The invention relates to the use of derivatives of 3-amidinophenylalanine as urokinase inhibitors for treating malignant tumors and the formation of metastases.

This application is a Continuation-in-part of U.S. Ser. No. 10/202,850filed Jul. 26, 2002, which is a divisional of U.S. Ser. No. 09/743,800filed Apr. 3, 2001, which is a 35 USC § 371 National Phase EntryApplication from PCT/EP99/05145 filed Jul. 20, 1999.

DESCRIPTION

The invention relates to the use of derivatives of3-amidinophenylalanine as urokinase inhibitors in particular fortreating malignant tumors and the formation of metastases or as agentsfor targeting lymphocytes and for treating disorders of the lymphatictissue, in particular lymphomas.

The ability of solid tumors to spread and metastasize in surroundingtissue correlates with the degradation or transformation of theextracellular matrix (tumor stroma) in the vicinity of the tumor celland/or with the ability of said tumors to penetrate the basementmembrane. Although the (patho)biochemical connections have not beencompletely elucidated yet, the plasminogen activator urokinase (uPA) andthe urokinase receptor (uPAR) play a central role. uPA mediates theproteolytic cleavage of plasminogen to give plasmin. Plasmin in turn isa protease which has a wide range of actions and is capable of directlybreaking down components of the extracellular matrix such as fibrin,fibronectin, laminin and the protein skeleton of proteoglycans. Inaddition, plasmin can activate “latent” metalloproteases and theinactive proenzyme of uPA, pro-uPA.

Tumor cells and non-malignant cells of the tumor stroma synthesize andsecrete the enzymatically inactive proenzyme pro-uPA. Proteases such as,for example, plasmin or the cathepsins B and L cleave pro-uPA by limitedproteolysis to give the active serine protease HMW-uPA (HMW=highmolecular weight). Pro-uPA and the active protease HMW-uPA bind to thecell surface receptor uPAR (CD87). Plasmin(ogen) likewise binds tospecific receptors on the plasma membranes of tumor cells which leads tofocused and amplified plasminogen activation in the immediate vicinityof the tumor cells. Invasive cells thus are able to break down theextracellular matrix without finding themselves deprived of the supportnecessary for directed movement because of proteolysis.

Various cytobiological studies have shown that the cell-associatedplasminogen activator system is of particular importance within thecascade-like reaction pathways of tumor-associated proteolytic systems(Wilhelm et al. (1994 The Urokinase/Urokinase receptor system: A newtarget for cancer therapy? In: Schmitt M., Graeff H., Kindermann G.(eds.): Prospects in Diagnosis and Treatment of Cancer. InternationalCongress Series, Excerpta Medica 1050, Amsterdam, Elsevier 1994, pp145-156). Cultures of human colon carcinoma cells showed that theirability to migrate through an extracellular matrix depended on thedegree of uPA receptor saturation with active uPA. (Hollas et al.,Cancer Res. 51 (1991), 3690-3695). The cell culture model likewiseshowed a reduction in the invasive potential of cells when PAI-1 (Cajotet al., Proc. Natl. Acad. Sci. USA 87 (1990), 6939-6943) or PAI-2 (Bakeret al., Cancer Res. 50 (1990), 4676-4684) inhibited the proteolyticactivity of uPA. A similar effect was achieved on inhibition of uPAbinding to the cell surface by blocking the receptor by means ofproteolytically inactive uPA variants (Cohen et al., Blood 78 (1991),479-487; Kobayashi et al., Br. J. Cancer 67 (0.1993), 537-544).Transfection of epidermoid carcinoma cells using a plasmid expressing anantisense transcript of a part of uPAR also reduced the invasivity ofsaid cells by suppressing uPAR synthesis (Kook, EMBO J. 13 (1994),3983-3991). Antibodies directed against uPA and PAI-1 reduced theinvasive potential of lung cancer cells in vitro (Liu et al., Int. J.Cancer 60 (1995), 501-506).

Animal models of tumors were also able to show the influence of theplasminogen activator system on the metastasizing process. Thus,addition of anti-uPA antibodies almost completely prevented theformation of lung metastases caused by human carcinoma cells in chickenembryos (Ossowski and Reich, Cell 35 (1983), 611-619). Metastasizinghuman carcinoma cells were transfected using an expression plasmid whichencoded a proteolytically inactive, but uPAR-binding uPA mutant. Themouse model showed that carcinoma cells synthesizing inactive uPAproduced a significantly smaller number of metastases after injectionthan nontransfected cells (Crowley et al., Proc. Natl. Acad. Sci. USA 90(1993), 5021-5025). Moreover, after administration of uPA antisenseoligonucleotides, nude mice showed inhibition of intraperitonealspreading of human ovarian carcinoma cells (Wilhelm et al., Clin. Exp.Metast. 13 (1995), 296-302).

In recent years, the clinical relevance of factors of the plasminogenactivator system (uPk, uPAR, PAI-1 and PA1-2) for the prognosis ofpatients having solid malignant tumors has been intensively studied. Inthese studies, the uPA antigen content in various tumors (e.g. breast,ovaries, stomach, lung, kidney) proved to be a strong prognostic factorboth for the recurrence-free survival and for the mortality (see forexample, Schmitt et al., J. Obstet. Gynaecol. 21 (1995), 151-165;Jaenicke et al., Breast Cancer Res. Treat. 24 (1993), 195-208; Kuhn etal., Gynecol. Oncol. 55 (1994), 401-409; Nekarda et al., Lancet 343(1994), 117; Pedersen et al., Cancer Res. 54 (1994), 4671-4675).Likewise, increased concentrations of uPAR in lung cancer tissue(Pedersen et al., supra) and breast cancer tissue (Duggan et al., Int.J. Cancer 61 (1995), 597-600; Ronne et al., Breast Cancer Res. Treat. 33(1995), 199-207) and also in the case of stomach cancer both in thetumor tissue itself (Heiss et al., J. Clin. Oncol. 13 (1995), 2084-2093)and in tumor cells disseminated into bone marrow (Heiss et al., NatureMedicine 1 (1995), 1035-1039) correlate with a poor prognosis.

The use of synthetic uPA inhibitors makes it possible to suppressinvasion and spreading of tumor cells. However, developing specific uPAinhibitors is difficult, since tissue plasminogen activator (tPA) has anidentical specificity for cleaving the peptide bond Arg560/Val561 ofplasminogen. In most cases therefore, low molecular weight uPAinhibitors also inhibit tPA and thus also tPA-mediated fibrinolysis. Inaddition, it must be guaranteed that synthetic uPA inhibitors show nostrong plasmin inhibition.

Despite these restrictions, some inhibitors are known which have acertain specificity for uPA, but a low inhibition capacity, such asbenzamidine derivatives and β-naphthamidine derivatives, the mosteffective compound inhibiting uPA with K_(i)=2.2 μmol/l (Stürzebecherand Markwardt, Pharmazie 33 (1978), 599), or amiloride with K_(i)=7μmol/l (Vassalli and Belin, FEBS. Lett. 214 (1987), 187-191).

DE-A-30 35 086 discloses cyclohexanecarboxylic acid derivatives whichhave inhibitory effects on proteases such as trypsin, chymotrypsin,thrombin or uPA. However, the compounds studied only show quite weakand, moreover, unspecific uPA inhibition. EP-A-0 183 271 discloseslysine derivatives and the use thereof as protease inhibitors. Abenzamidinolysine derivative (compound 108) is also described whichinhibits uPA in vitro, but acts comparably on other proteases such astrypsin or plasma kallikrein. WO 95/17885 disloses low molecular weightpolypeptides as uPA inhibitors.

Another class of known uPA inhibitors is represented by 4-substitutedbenzothiophene-2-carboxamidines with K_(i)=0.16 mmol/l in the case ofbenzothiophene 623 (Towle et al., Cancer Res. 53 (1993), 2553-2559).These inhibitors have a significantly higher affinity for uPA than fortPA and plasmin. uPAR-bound uPA, too, is inhibited very effectively.Disadvantageously however, the chemical synthesis of these substances iscomplicated and few possibilities for structural modifications arepresent or have been demonstrated until now.

Therefore, the development of further uPA inhibitors is very beneficialfor further elucidating the role of uPA and uPAR in various diseases,especially in tumor spreading and metastasizing.

Nα-Arylsulfonyl and Nα-arylsulfonylaminoacyl derivatives of3-amidinophenylalanine are known as selective inhibitors of thrombin(Markwardt et al., Thromb. Res. 17 (1980), 425-431) or of coagulationfactor Xa (Stürzebecher et al., Thromb. Res. 54 (1989), 245-252). WO92/08709, WO 94/18185 and WO 96/05189 also disclose the use ofamidinophenylalanine derivatives as inhibitors of blood clotting, inparticular as inhibitors of thrombin.

Piperidides and piperazides of 3-amidinophenylalanine have beenintensively studied, among which lead structures for inhibitingfibrinolytic enzymes have been found (Stürzebecher et al., J. EnzymeInhibition 9, 87-99, 1995; Stürzebecher et al., J. Med. Chem. 40,3091-3099, 1997). While Stürzebecher et al. (1995) merely describeinhibition of thrombin, factor Xa, plasmin and trypsin, Stürzebecher etal. (1997) also provide information about inhibiting uPA.Nα-2-Naphthylsulfonyl-,Nα-2-(2,2,5,7,8-pentamethylchroman-6-yl)sulfonyl- andNα-2-camphor-10-yl-sulfonyl-substituted3-amidinophenylalaninepiperazides have a K_(i) for uPA of from 28 to 140μmol/l, which is about three orders of magnitude higher than theinhibition constant for thrombin. Thus it was impossible to assume that3-amidinophenylalanine derivatives are suitable as urokinase inhibitors.

Surprisingly we have found, however, that 3-amidinophenylalaninederivatives substituted in the 2 position by a phenyl radical representselective uPA inhibitors which are active in vivo. Furthermore, we havefound that these substances have high selectivity for lymphatic tissueand thus are suitable as agents for targeting lymphocytes, for examplefor treating malignant disorders of the lymphatic tissue such aslymphomas.

The present invention relates to novel urokinase inhibitors of thegeneral formula I,

which are derived from 3-amidinophenylalanine and are present asracemates and also as L- or D-configured compounds and in which

-   R¹ (a) is OH or OR⁴, where R⁴ is unsubstituted or substituted, for    example by hydroxyl, carboxyl, sulfonyl, nitro, cyano, oxo and/or    halogen, branched or unbranched C₁-C₈-alkyl, C₃-C₈-cycloalkyl or    aralkyl, e.g. benzyl or phenylethyl,    -   (b) represents a group of the formula

-   -    in which R⁵ and R⁶ are any radicals compatible with the overall        structure, where in particular        -   (i) R⁵ and R⁶ are H,        -   (ii) R⁵ is H and R⁶ is unsubstituted or substituted, for            example by hydroxyl, carboxyl, sulfonyl, nitro, cyano, oxo            and/or halogen, branched or unbranched C₁-C₈-alkyl, aralkyl,            e.g. benzyl or phenylethyl, or C_(s)-C₈-cycloalkyl,        -   (iii) R⁵ and R⁶ are in each case independently unsubstituted            or substituted, for example by hydroxyl or/and halogen,            unbranched or branched C₁-C₄-alkyl or        -   (iv) R⁵ is H and R⁶ is —NH₂ or is, in particular, an            aryl-substituted or heteroaryl-substituted amino group,        -   (v) R⁵ is H or unsubstituted or substituted, for example by            hydroxyl or/and halogen, unbranched or branched C₁-C₄-alkyl,            and R⁶ is an amino acid residue, for example an α-, β- or            ω-amino carboxylic acid or amino sulfonic acid residue, a            peptide residue, for example of up to 50 amino acids in            length, or a polypeptide residue, for example of from            greater than 50 amino acids to 1000 amino acids in length,    -   (c) represents a group of the formula

-   -    in which m is the number 1 or 2 and in which one or more of the        methylene groups are optionally substituted, for example by        hydroxyl, carboxyl, C₁-C₄-alkyl or aralkyl, e.g. benzyl or        phenylethyl, with the group (c) being racemic or in D or L        configuration, and R⁷ has the meaning of R¹ in subsections        (a), (b) and (f),    -   (d) represents a group of the formula

-   -    in which p=r=1, p=1 and r=2 or p=2 and r=1 and in which one or        more of the methylene groups are optionally substituted, for        example by hydroxyl, carboxyl, C₁-C₄-alkyl or aralkyl, e.g.        benzyl or phenylethyl, and R⁷ has the meaning of R¹ in        subsections (a), (b) and (f),    -   (e) represents a piperidyl group which is unsubstituted or        substituted in one of positions 2, 3 or 4, for example by        C₁-C₄-alkyl, C₁-C₃-alkoxy or hydroxyl,    -    where a further aromatic or cycloaliphatic ring, preferably        phenyl or cyclohexyl, is optionally fused to the        heterocycloaliphatic rings of the formulae (c), (d) and (e) in        the 2,3 position or the 3,4 position relative to the heteroatom,    -   (f) represents a group of the formula

-   -    in which R⁸ is        -   (i) unsubstituted or, for example, C₁-C₄-alkyl-,            C₁-C₃-alkoxy-, hydroxyl-, carboxyl, sulfonyl-, nitro-,            cyano-, oxo- or/and halogen-substituted C₁-C₆-alkyl or aryl,            such as, for example, phenyl, p-halophenyl or naphthyl,        -   (ii) saturated or unsaturated, branched or unbranched            C₁-C₆-alkoxy or        -   (iii) unsubstituted or, for example, C₁-C₆-alkyl-,            C₁-C₃-alkoxy-, hydroxyl-, carboxyl-, sulfonyl-, nitro-,            cyano-, oxo- or/and halogen-substituted phenoxy or            benzyloxycarbonyl,    -   (g) represents an acyl radical of the formula —COX, where X is        -   (i) H or unsubstituted, for example hydroxyl-, carboxyl-,            sulfonyl-, nitro-, cyano-, oxo- or/and halogen-substituted,            unbranched or branched alkyl, preferably C₁-C₆-alkyl, in            particular methyl,        -   (ii) unsubstituted or, for example, C₁-C₆-alkyl-,            C₁-C₃-alkoxy-, hydroxyl-, carboxyl-, sulfonyl-, nitro-,            cyano-, oxo- or/and halogen-substituted aryl or heteroaryl,            such as, for example, phenyl, p-halophenyl or thienyl, or        -   (iii) unsubstituted or, for example, hydroxyl-, carboxyl-,            sulfonyl-, nitro-, cyano-, -oxo- or/and halogen-substituted            cycloalkyl, preferably C₃-C₁₀-cycloalkyl,    -   (h) represents aralkyl, e.g. benzyl or phenylethyl, in which the        aromatic radical is unsubstituted or substituted, for example by        halogen, C₁-C₆-alkyl, C₁-C₃-alkoxy, hydroxyl, cyano, carboxyl,        sulfonyl or nitro,    -   (i) represents a carboxamide radical of the formula —CONR′R″ a        thiocarboxamide radical —CSNR′R″, or an acetamide radical        —CH₂—CONR′R″        -   where        -   (i) R′ and R″ are H,        -   (ii) R′ and R″ are in each case independently C₁-C₄-alkyl,        -   (iii) R′ is H and R″ is C₁-C₄-alkyl,        -   (iv) R′ is H and R″ is aryl, e.g. phenyl, or        -   (v) R′ and R″ constitute together with the nitrogen atom a            heterocycloaliphatic ring having 5-7 ring members and            possibly having a further heteroatom, e.g. N, O or/and S,    -   (j) represents SO₂—Y where Y is        -   (i) unsubstituted or, for example, hydroxyl-, carboxyl-,            sulfonyl-, nitro-, cyano-, oxo- or/and halogen-substituted            C₁-C₈-alkyl, preferably methyl, trifluoromethyl,            trichloromethyl,        -   (ii) unsubstituted or, for example, C₁-C₆-alkyl-,            C₁-C₃-alkoxy-, hydroxyl-, carboxyl-, sulfonyl-, nitro-,            cyano-, oxo- or/and halogen-substituted aryl or heteroaryl,            such as, for example, phenyl, 4-methylphenyl,            2,4,6-trimethylphenyl, 2,4,6-triisopropylphenyl,            4-methoxy-2,3,6-trimethylphenyl,            2,2-dimethyl-6-methoxy-chromanyl,            2,2,5,7,8-pentamethylchromanyl, anthraquinonyl, naphthyl or            quinolyl, or O-aryl, preferably O-phenyl or O-heteroaryl or        -   (iii) —NR′R″, where R′ and R″ are in each case independently            H or C₁-C₃-alkyl,    -   (k) represents a cycloaliphatic ring having from 5 to 8 carbon        atoms, which is unsubstituted or substituted, for example by        C₁-C₆-alkyl, C₁-C₃-alkoxy, halogen, hydroxyl or/and oxo,    -   (l) represents an unsubstituted or, for example, C₁-C₆-alkyl-,        C₁-C₃-alkoxy-, hydroxyl-, carboxyl-, sulfonyl-, nitro-, cyano-,        oxo- or/and halogen-substituted heteroaryl radical such as, for        example, pyridyl or pyrimidyl, or heterocycloaliphatic radical,        for example N-methylpiperidyl,    -   (m) represents a functionalized alkyl radical of the formula        —(CH₂)_(n)—X, where the alkyl chain is unbranched or branched,        n=1 to 8, and the functional radical X        -   (i) represents a hydroxyl group whose hydrogen atom is            unsubstituted or substituted by C₁-C₄-alkyl, aralkyl, e.g.            benzyl or phenylethyl, aryl, e.g. phenyl, C₁-C₄-hydroxyalkyl            or acyl group Co-alkyl, (C₁-C₆)        -   (ii) is a halogen atom,        -   (iii) represents a tertiary amino group of the formula            —N(alk)₂, where the alkyl groups have 1 to 3 carbon atoms            and are preferably the same, and the nitrogen atom may            belong to a heterocycloaliphatic ring having 5-7 ring            members and possibly having a further heteroatom, e.g. N, O            or/and S,

-   R² represents unsubstituted or, for example, C₁-C₆-alkyl-,    C₁-C₃-alkoxy-, hydroxyl-, carboxyl-, sulfonyl-, nitro-, cyano-, oxo-    or/and halogen-substituted phenyl, such as, for example, phenyl,    4-methylphenyl, 2,4,6-trimethylphenyl, 2,4,6-triisopropylphenyl,    4-methoxy-2,3,6-trimethylphenyl,

-   R³ is H or branched or unbranched C₁-C₄-alkyl, and n is 0 or 1.

The compounds may also be present as salts, preferably asphysiologically acceptable acid salts, for example as salts of mineralacids, particularly preferably as hydrochlorides, or as salts ofsuitable organic acids.

Of the compounds defined in the general claims, those are of particularimportance in which R¹ corresponds to a group of the formulae (b), (d)and (f), R² represents phenyl mono-, di- or trisubstituted by alkyl, inparticular 2,4,6-substituted phenyl, e.g. 2,4,6-triisopropylphenyl, andn=0.

It is possible to prepare the compounds of the general formula I in amanner known in principle, for example as described in WO 92/08709 andWO 94/18185, and to assay their biological in vitro activity.

(L)-, (D) or (D,L)-3-cyanophenylalanine methyl ester hydrochloride isreacted with an appropriate sulfonyl chloride or a sulfonated amino acidor the halide thereof in the presence of a base to give a compound ofthe general formula I, which has a cyano function and in which R¹=OCH₃and R² and also R³ and n correspond to the meanings defined in thegeneral claims. Mild acidic or alkaline hydrolysis produces therefromthe compounds of the general formula I, which have carboxylic acidstructure (R¹=—OH) and whose acid-catalyzed esterification with anappropriate alcohol leads to compounds of the general formula I, whereR¹=(a). Applying a method common in peptide chemistry, for example DCCin the presence of HOBt, reacting the carboxylic acids of the generalformula I (R¹=OH) with a nucleophile of the structures (b), (e) and (f)may give compounds with the corresponding R¹ of the general formula I.To synthesize compounds with R¹=(c) and (d), carboxylic acids of thegeneral formula I with R¹=OH are first reacted with cycloaliphatic aminoacid esters of the structures (c) and (d), where R⁷ is preferably —OCH₃or OC₂H₅, the carboxylic esters obtained are hydrolyzed under mildacidic or alkaline conditions to give the corresponding carboxylic acidswhich may subsequently be esterified in a manner already described or bereacted with nucleophiles of the structures (b), (e) and (f), andcompounds of the general formula I with R¹=(c) and also (d) and withR⁷=(a), (b), (e) and (f) are obtained.

The target compounds of the general formula I, which have amidinestructure, are obtainable from the cyano compounds in a known manner;normally, the thioamides are obtained first by addition of H₂S to thecyano group, and are converted by S-methylation with methyl iodide intothe thioimido esters and then into the amidino compounds by treatmentwith ammonium acetate in alcoholic solution. In addition and whereappropriate, it is possible, using methanol or ethanol in the presenceof HCl gas and, in particular cases, of an inert solvent, to preparefrom the cyano compounds the corresponding imido ester hydrochlorides,which are reacted in alcoholic ammonia solution to give the amidinocompounds.

The urokinase inhibitors according to the invention may be used, whereappropriate, together with at least one suitable pharmaceuticalexcipient or carrier for producing orally, subcutaneously orintravenously administrable medicaments for controlling tumors or fordiagnosis. Likewise possible is administration in combination with otheractive substances, for example other urokinase inhibitors such asantibodies or/and peptides.

The medicaments for controlling tumors in humans and animals may beadministered topically, orally, rectally or parenterally, e.g.subcutaneously or intravenously, in the form of tablets, coated tablets,capsules, pellets, suppositories, solutions or transdermal systems suchas plasters.

A particularly preferred compound of the formula (I) isNα-(2,4,6-triisopropylphenylsulfonyl)-3-amidino-(D,L)-phenylalanine4-ethoxycarbonylpiperazide hydrochloride or the L enantiomer thereof ora pharmaceutically suitable salt of these compounds. These substanceshave good solubility. They are soluble in Tris buffer (pH 7.3) up to aconcentration of 5×10⁻⁵ mol/l. Addition of 5% ethanol increases thesolubility to 2×10⁻⁴ moll and addition of 5% DMSO to 10⁻³ mol/l.

The compounds of the invention are capable of very effectivelyinhibiting the growth or/and spreading of malignant tumors, for exampletumor spreading of pancreatic carcinoma, tumor growth of breastcarcinoma and also metastasizing of tumors. It is possible to use theuPA inhibitors, where appropriate, together with other anti-tumor agentsor with other types of treatment, e.g. radiation or surgery.Furthermore, the inhibitors according to the invention are alsoeffective in other uPA-associated disorders (e.g. in preventingformation of blisters in the case of the skin disorder pemphigusvulgaris).

uPA inhibitors according to the invention are preferably characterizedin that they have a K_(i) which is at least twofold, preferably at least5-fold and particularly preferably at least 10-fold lower for uPA thanfor tPA. It is furthermore remarkable that the compounds of theinvention only marginally affect blood clotting, since their K_(i) istoo high for effective inhibition of thrombin and factor Xa.

The inventive substances of the formula I may be used in the form ofconjugates with physiologically active substances, for example withradiolabels or cytotoxic agents, e.g. chemotherapeutics such ascisplatin or 5-fluorouracil, or with peptides. Furthermore it ispossible to incorporate the substances into the membrane of carriervesicles, e.g. liposomes, and thus to facilitate targeting of activesubstances enclosed in the carrier vesicles, for example cytotoxicagents such as doxorubicin.

Another indication for the substances of the general formula II:X—R²,where X is any radical, in particular an organic radical, for example aradical as defined for compounds of the formula I, but also anotherradical, for example a physiologically active substance such as acytotoxic agent, a peptide or a radiolabel, a lipid or a carbohydrate,and R² is a group as defined above, in particular 2,4,6-trisubstitutedphenyl, e.g. 2,4,6-triisopropylphenyl, is the targeting of lymphocytes,which is possible owing to a 10- to 20-fold higher affinity of saidsubstances for lymph node tissue than for other types of tissue. R² ispreferably linked to the radical X via an —SO₂— sulfonyl group. Thus,these substances are excellently suited as diagnostic agents or asagents for treating diseases of the lymphatic tissue, in particularmalignant diseases such as tumor metastases and lymphomas. Administeringthe substances may be carried out as already described above. Diseasesof the lymphatic tissue are preferably treated by administering themedicament over a number of days, for example over a period of from 5 to20 days, followed by a treatment break and, where appropriate, by one ormore administration repeats.

The following examples and figures are intended to illustrate theinvention in more detail. In the figures:

FIG. 1 depicts the result from determining the cytotoxicity of asubstance of the invention,

FIG. 2 depicts the experimental result from inhibiting the degradationby human breast carcinoma cells of a fibrin matrix,

FIGS. 3 and 4 depict the effect of a substance of the invention on thespreading, growth and metastasizing of breast carcinoma cells in rats,

FIG. 5 depicts the effect of a substance of the invention on the growthof a pancreatic tumor in rats, and

FIG. 6 depicts the effect of a substance of the invention on the growthof human breast carcinoma cells in mice.

FIG. 7 depicts the anti-metastatic efficacy ofNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide in a setting implying surgical resection ofthe orthotopically growing tumor at a defined size and various timepoints of therapy onset relative to resection.

FIG. 8 displays the results of a BN472 therapy study aiming atestablishing efficacy ofNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide at prolonged dosing intervals.

FIG. 9 shows additive anti-metastatic benefits ofNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide in the BN472 cancer model in combination withstandard cytotoxic anti-tumor therapies epirubicin or 5-fluorouracil(5-FU).

FIG. 10 shows the time profiles of mean plasma concentrations ofNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine-4-ethoxycarbonylpiperazideat the end of a 30 min. infusion period in human patients.

FIG. 11 shows the effect of epirubicin;Nα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide and epirubicin; andNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide on BN-472 mammary tumor load in Brown Norwayrats.

FIG. 12 shows the effect of epirubicin;Nα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide and epirubicin; andNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide on BN-472 mammary tumor weight in BrownNorway rats.

FIG. 13 shows the effect of epirubicin;Nα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide and epirubicin; andNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide administrations on axillary lymph-nodeweight.

FIG. 14 shows the effect of epirubicin;Nα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide and epirubicin; andNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide administrations on interperitoneal lymph nodeweight.

FIG. 15 shows the effect of epirubicin;Nα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide and epirubicin; andNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide administrations on the number of macroscopiclung foci.

FIG. 16 shows the effect of paclitaxel;Nα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide and paclitaxel; andNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide administrations on axillary lymph-nodeweight.

FIG. 17 shows the effect of paclitaxel;Nα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide and paclitaxel; andNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide administrations on interperitoneal lymph nodeweight.

FIG. 18 shows the effect of paclitaxel;Nα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide and paclitaxel; andNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide administrations on the number of macroscopiclung foci.

FIG. 19 shows the effect of 5-FU;Nα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide and 5-FU; andNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide on BN-472 mammary tumor growth in BrownNorway rats.

FIG. 20 shows the effect of 5-FU;Nα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide and 5-FU; andNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide on BN-472 mammary tumor burden in BrownNorway rats.

FIG. 21 also shows the effect of 5-FU;Nα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide and 5-FU; andNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide on BN-472 mammary tumor burden in BrownNorway rats.

FIG. 22 shows the anti-metastatic effects of 5-FU;Nα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide and 5-FU; andNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide in Brown Norway rats bearing BN-472 mammarytumors.

FIG. 23 also shows the anti-metastatic effects of 5-FU;Nα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide and 5-FU; andNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide in Brown Norway rats bearing BN-472 mammarytumors.

FIG. 24 also shows the anti-metastatic effects of 5-FU;Nα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide and 5-FU; andNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine4-ethoxycarbonylpiperazide in Brown Norway rats bearing BN-472 mammarytumors.

EXAMPLES 1.Nα-2,4,6-Triisopropylphenylsulfonyl-(L)-3-amidinophenylalanine4-ethoxycarbonylpiperazide hydrochloride 1.1Nα-2,4,6-Triisopropylphenylsulfonyl-(L)-3-cyanophenylalanine MethylEster

5 g of (L)-3-cyanophenylalanine methyl ester were suspended in 100 ml ofdioxane, 4.45 ml of N-methylmorpholine (NM) were added and the mixturewas stirred for 30 min. After adding 5.97 g of2,4,6-triisopropylbenzenesulfonyl chloride in solid form and subsequentstirring for 3 days, precipitated NMM-HCl was filtered off, the solventwas distilled off and the crude product obtained was purified on silicagel (SG) 60 (chloroform). Yield: 8.34 g of syrup (90%).

1.2 Nα-2,4,6-Triisopropylphenylsulfonyl-(L)-3-cyanophenylalanine

8.34 g of compound 1.1 were heated under reflux in a mixture of 50 mleach of acetic acid and 1 N hydrochloric acid for 8 h and, aftercooling, extracted twice with ethyl acetate; the combined ethyl acetatesolutions were dried over MgSO₄ and the solvent was distilled off. Afterpurification on SG 60 (chloroform), 5.8 g of a solid product wereobtained (72%).

1.3 Nα-2,4,6-Triisopropylphenylsulfonyl-(L)-3-cyanophenylalanine4-ethoxycarbonylpiperazide

5.7 g of compound 1.2 were dissolved in 100 ml of tetrahydrofuran (THF)and cooled to 0° C.; 2.22 g of α-hydroxybenzotriazole (HOBt) and 2.82 gof dicyclohexylcarbodiimide (DCC) were added and the mixture was stirredfor 30 min. After adding 3.94 g of 1-ethoxycarbonylpiperazine in 30 mlof THF and subsequent stirring overnight, precipitated dicyclohexylurea(DCU) was filtered off, the solvent was distilled off and the crudeproduct obtained was purified on SG 60 (chloroform). Yield: 7.1 g of anamorphous powder (96%).

1.4. Nα-2,4,6-Triisopropylphenylsulfonyl-(L)-3-amidinophenylalanine4-ethoxycarbonylpiperazide Hydrochloride

7.1 g of compound 1.3 were dissolved in 30 ml of pyridine, 30 drops oftriethanolamine (TEA) were added, a vigorous stream of hydrogen sulfidewas introduced for 10 min, and the mixture was left at room temperaturefor 2 days. The solvent was then distilled off, the residue wasdissolved in ethyl acetate, the organic phase was washed with 1 Nhydrochloric acid and saturated sodium chloride solution and dried overMgSO₄, and the solvent was distilled off. 7.2 g of thioamide obtained inthis way were dissolved in 250 ml of acetone, 17 g of methyl iodide wereadded to the solution, and the mixture was left at room temperatureunder protection from light for 2 days. The solvent was then distilledoff, the thioimido ester hydroiodide (8.5 g) was dissolved in 50 ml ofmethanol, 1.9 g of ammonium acetate were added and the mixture washeated to 60° C. for 4 h. After distilling off the solvent, the crudeproduct obtained was purified on Sephadex LH20 (methanol). The amidinehydroiodide obtained in this way was converted into the hydrochloridevia an ion exchange column (Amberlite IRA-420). Yield: 5.3 g of anamorphous powder (69%).

2Nα-2,4,6-triisopropylphenylsulfonyl-(D,L)-3-amidinophenylalanylnipecoticacid Benzylamide Hydrochloride 2.1 EthylNα-2,4,6-triisopropylphenylsulfonyl-(D,L)-3-cyanophenylalanylnipecotate

4.56 g of Nα-2,4,6-triisopropylphenylsulfonyl-(D,L)-3-cyanophenylalanine(prepared from (D,L)-3-cyanophenylalanine methyl ester hydrochloride andthe appropriate sulfonyl chloride analogously to 1.1 and 1.2), 1.5 g ofHOBt and 2.42 g of DCC were dissolved in 50 ml of DMF; the mixture wasstirred for 1 h, and then 2.36 g of ethyl nipecotate were added. Afterstirring overnight, precipitated DCU was filtered off, the solvent wasdistilled off and the residue was dissolved in a small amount ofmethanol and left to crystallize. The precipitate formed was filteredoff with suction, washed with methanol and dried. Yield: 4.46 g (75%).

2.2Nα-2,4,6-Triisopropylphenylsulfonyl-(D,L)-3-cyanophenylalanylnipecoticAcid

4.4 g of the above-described ethyl ester were heated under reflux in amixture of 35 ml of acetic acid and 25 ml of 1 N HCl for 2 h. Afteradding 10 ml of water, the mixture was left to cool, and a wax-likeproduct precipitated. After decanting the solvent, 200 ml of water wereadded, the mixture was stirred over a relatively long period, and thesolid substance obtained was filtered off with suction, washed withwater and dried. Yield: 3.84 g (92%).

2.3Nα-2,4,6-triisopropylphenylsulfonyl-(D,L)-3-cyanophenylalanylnipecoticAcid Benzylamide

2.28 g of the above-described compound, 0.6 g of HOBt and 0.97 g of DCCwere dissolved in 20 ml of DMF and the mixture was stirred for 1 h; 0.6g of benzylamine was then added, and stirring continued overnight. Afterfiltering off the precipitated DCU, the solvent was distilled off, theresidue was dissolved in methanol and the solution was poured into 5%strength sodium hydrogen carbonate solution/ice. After 1 h, theprecipitate formed was filtered off with suction, washed with water anddried in vacuo. Yield: 2.48 g (94%).

2.4Nα-2,4,6-triisopropylphenylsulfonyl-(D,L)-3-amidinophenylalanylnipecoticAcid Benzylamide Hydrochloride

2.4 g of compound 2.3 were dissolved in 30 ml of pyridine, 30 drops oftriethanolamine (TEA) were added, a vigorous stream of hydrogen sulfidewas introduced for 10 min, and the mixture was left at room temperaturefor 2 days. The solvent was then distilled off, the residue wasdissolved in ethyl acetate, and extracted with 1 N hydrochloric acid.After washing the organic phase with saturated sodium chloride solutionand drying over sodium sulfate, the solvent was distil ed off. 2.38 g ofthe thioamide obtained in this way were dissolved in 100 ml of acetone,6.5 g of methyl iodide were added to the solution, and the mixture wasleft at room temperature under protection from light for 20 h. Thesolvent was then distilled off, the thioimido ester hydroiodide wasdissolved in 50 ml of methanol, 0.5 g of ammonium acetate was added andthe mixture was heated to 60° C. in a water bath for 4 h. Afterdistilling off the solvent, the crude product obtained was purified onSG 60. Elution was carried out first with chloroform, then withchloroform/methanol 9:1. The amidine hydroiodide obtained in this waywas converted into the hydrochloride on an ion exchange column(Amberlite IRA-420). Yield: 1.45 g of an amorphous powder (56%).

The compounds were characterized using mass spectrometry, and purity waschecked by means of TLC and HPLC.

3. In Vitro Inhibition of Urokinase by Selected Compounds of the FormulaI

Configuration R¹ R² n Ki, μmol/l L —N N—COOC₂H₅ TIPP 0 0.41 D,L —NN—COOC₂H₅ TIPP 0 0.96 Abbreviations: TIPP - 2,4,6-triisopropylphenylDetermination of Inhibition Activity

To determine the inhibitory activity, 200 μl of Tris buffer (0.05 mol/l,containing the inhibitor, 0.154 mol/l NaCl, 5% ethanol pH 8.0), 25 μl ofsubstrate (Pefachrome UK or Bz-βAla-Gly-Arg-pNA in H₂O; Pentapharm Ltd.,Basle, Switzerland) and 50 μl of sc-urokinase (Ribosepharm GmbH, Haan,Germany) were incubated at 25° C. After 3 min the reaction was stoppedby adding 25 μl of acetic acid (50%), and the absorption at 405 nm wasdetermined by means of a Microplate Reader (MR 5000, Dynatech,Denkendorf, Germany). K_(i) values were determined according to Dixon bylinear regression using a computer program. The K_(i) values are themean values of at least three determinations with a standard deviationof less than 25%.

4. In Vitro Inhibition of Various Serine Proteases of the Trypsin Typeby (Nα-2,4,6-triisopropylphenylsulfonyl-(L)-3-amidinophenylalanine4-ethoxycarbonylpiperazide (uPA Inhibitor) Compared withNα-2-naphthylsulfonyl-3-amidinophenylalanine N′-methylpiperazide(Naphthylsulfonyl Derivative)

Ki [μmol/l] Naphthylsulfonyl Enzyme uPA-Inh. derivative Urokinase 0.41150 Plasmin 0.39 55 Sc-tPA 4.9 430 Thrombin 0.49 0.036 Factor Xa 1.7 30Factor XIIa 13 >1000 Plasma kallikrein 7.2 85 Glandularkallikrein >1000 >1000 Trypsin 0.037 1.3 Tryptase 6.3 33

The inhibition activities of the enzymes used were determined accordingto the principle described in Example 3.

The values given above indicate that the uPA inhibitor according to theinvention has a K_(i) for urokinase which according to the invention hasa K_(i) for urokinase which is more than ten times smaller than theK_(i) for single chain tPA (Sc-tPA). Thus, the substances of theinvention are suitable as selective urokinase inhibitors. Forcomparison, the inhibitory activity of the naphthylsulfonyl derivativeis given which has a significantly lower in vitro anti-uPA activity.

5. Cytotoxicity Determination

To determine cell proliferation/cytotoxicity a commercially availabletest was used (Promega) which is based on the cellular conversion of atetrazolium salt. The colored product resulting from this reaction canbe quantified by means of an ELISA spectrometer (ICN flow). Thesynthetic inhibitor (open circles) had no effect on the growth of thehuman ovarian carcinoma cells OV-MZ-6 (FIG. 1) when compared with thesolvent alone (closed circles). Thus, the inventive uPA inhibitor is notcytotoxic in pharmacologically effective concentrations up to 40 μM.

6. Inhibition of the Degradation by Human Breast Carcinoma Cells of aFibrin Matrix

To study the potential of tumor cells for breaking down an extracellularmatrix, a fibrin matrix degradation assay was developed and used. Agreater proteolytic activity of the tumor cells leads to a higherconcentration of fibrin degradation products in the matrix supernatant.The matrix degradation capacity corresponds to the concentration offibrin degradation products which are determined by means of ELISA (Ddimer).

The fibrin gels were prepared in 24-well culture dishes from 200 ml offibrinogen (50 mg/ml) in PBS (pH 7.4), by 50 μl of thrombin (10 U/ml)and 50 μl of CaCl₂ (150 mM) per well after incubation at 37° C. for 30minutes. 2×10⁵ breast carcinoma cells were seeded on said fibrin matrixin 1 ml of DMEM culture medium plus 10% fetal calf serum and 2 μg ofGlu-plasminogen, and incubated for 4 h. The supernatant was thencentrifuged, in order to remove the cells, and the fibrin degradationproducts were quantified by means of ELISA. Adding the inhibitor (A) atdifferent concentrations caused significant inhibition of matrixdegradation by breast carcinoma cells compared with the naphthylderivative (B) which shows no inhibition of fibrin degradation by breastcarcinoma cells (FIG. 2).

7. In Vitro Assay of the uPA Inhibitor for Tumor Spreading, Tumor Growthand Metastasizing in Rats

A) Breast Cancer Model

10-25 mm³ of BN-472 breast cancer tumor fragments from rats weretransplanted into female brown Norwegian rats from 6 to 7 weeks old,subcutaneously and orthotopically under the subcutaneous fat of themammary gland (Day 0). The treatment of the animals was startedintraperitoneally 24 hours after tumor inoculation. Each group consistedof eight animals. The control group received only the injection solution(100 μl of a 10% ethanol/saline (0.9% NaCl) solution). A dose of 1 mg/kgbody weight was intraperitoneally administered on a daily basis to thecomparative group of the naphthyl derivative. (B) and to the therapygroup of the inventive uPA inhibitor (A). The treatment was carried outover a period of 4 weeks.

The dimensions of the subcutaneous tumors and the weight of the animalswere determined weekly. At the end of the treatment the animals weresacrificed and tumor weights, organ weights and the number of metastasesin relevant tissues were determined.

Treatment with uPA inhibitor (A) resulted in a significant reduction inthe weight of the primary tumor and also of the axillary lymph nodes(p=0.003 and p=0.005) compared with the naphthyl derivative (B) andcontrol groups without inhibitor (FIGS. 3 and 4). The weights of lung,liver, kidney and spleen were unchanged in the animals treated with theuPA inhibitor compared to the control animals.

B) Pancreatic Carcinoma Model

Fragments of the transplantable and metastasizing pancreaticadenocarcinoma CA20948 from rats were explanted from donor animals.After cell isolation, equal amounts of suspended tumor cells togetherwith 2 mg of Matrigel were subcutaneously implanted into each of theacceptor animals, male 10 week-old Lewis rats (n=9). The treatmentprocedure and also the composition of the therapy groups were the sameas under A).

FIG. 5 shows for the inventive uPA inhibitor (open circles) asignificant reduction in tumor weight and a decrease in the growth ofdeveloping rat pancreatic carcinomas compared to the naphthyl derivative(closed circles) and the control group (triangles).

C) Repeat of the Experiments with Different Mode of Administration

The experiments described in sections A and B were repeated with theinhibitor administered in a different way. For this, the inhibitor wasadministered subcutaneously in the breast carcinoma model (n=9) andintraperitoneally in the pancreatic adenocarcinoma model (n=8) withoutchanging the daily dose. The results of these repeat experimentscorresponded to the results already discussed, both in tendency andextent.

D) Summary of the Results

Treatment with the inhibitor achieved in all experiments a considerablereduction in tumor size and tumor weight and in the number and mass ofmetastases in comparison with the control groups. In theinhibitor-treated group of the breast tumor model, the average tumorweights at the end of the treatment were reduced to 23% (i.p.) or 37%(s.c.) compared to the vehicle-treated control. The number of lung fociin inhibitor-treated groups was reduced to 9% (i.p.) or 32% (s.c.) andthe mean weights of the axillary lymph nodes to 27% (i.p.) or 48%(s.c.).

In the inhibitor-treated groups of the pancreatic tumor model, the massof the tumor-containing pancreas was reduced by 76% (i.p.) or 34%(s.c.), and the masses of the subcutaneous tumors by 54% (i.p.) or 60%(s.c.), compared to the respective vehicle-treated groups. The number ofdetected liver foci in inhibitor-treated groups was 29% (i.p.), or 2%(s.c.), compared to the vehicle-treated control groups.

The development of the body weight increase and comparison of the organweights between inhibitor-treated and vehicle-treated groups showed noindications of any considerable toxicity of the inhibitor under thedescribed conditions.

8. Treatment of Human Breast Cancer Cells in Nude Mice

In order to test the in vivo efficacy of the inhibitor for inhibitingtumor growth of human breast carcinoma cells (MDA-BA-231), 6×10⁶ cellswere injected subcutaneously into the right flank of Balb/c nude mice(4-6 weeks old). The tumor cells were preincubated with the syntheticuPA inhibitor prior to inoculation. After 24 h, the mice were treatedtwice a week intraperitoneally with a dose of 1.2 mg/kg body weight asdescribed under A). The tumor size was determined weekly by measuringthe two largest diameters.

FIG. 6 shows that the tumor volume increases significantly more slowlyon administration of the uPA inhibitor (open circles) than in thecontrol group (closed circles) in which ethanol in saline wasadministered.

9. Biodistribution of the Inhibitor in Rats

The biodistribution of the inhibitor was determined by two independentexperiments in tissue extracts of rats which had been treated once a daywith 1 mg/kg i.p. of inhibitor i.p. over a period of 5 or 10 days. Forthe lysis, in each case 100 mg of tissue were mechanically comminutedand mixed with 200 μl of 1% Triton X-100 in physiological NaCl solution.After adding 400 μl of ethanol, the mixture was sonicated for 1 min. Thetissue extract was centrifuged at 12,000×g for 15 min. Forprepurification, the supernatant was applied to a C18 Silica ReversedPhase column (Sep-Pak® cartridge C18, 1 ml Water, Eschborn, Germany),equilibrated with 1 ml of methanol and 1 ml of water, washedsuccessively with 2×1 ml H₂O, 1 ml 10% methanol, 1 ml H₂O, 1 ml 5%acetonitrile, 0.04% perchloric acid and 1 ml H₂O and eluted with 500 μlof 75% acetonitrile, 0.04% perchloric acid. HPLC analysis was carriedout on a reversed phase C18 silica column with a 5-55% strengthacetonitrile gradient containing 0.04% perchloric acid.

The concentrations of the inhibitor and, for 5 comparison, of acorresponding naphthylsulfonyl derivative in each of the tissue typesstudied (μg/g) and also in blood plasma and bile (μg/ml) are depicted inthe following table.

TABLE Distribution profile of a substance of the invention in varioustissues and in blood plasma and bile in comparison with anaphthylsulfonyl derivative (Nα-2-naphthylsulfonyl-3-amidinophenylalanine 4-ethoxy- carbonylpiperazide uPAuPA inhibitor inhibitor Naphthylsulfonyl 1 mg/kg 1 mg/kg derivative i.p.i.p. 10 1 mg/kg i.p. Tissue 5 days days 5 days Content (μg/g) Spleen2.02 2.48 0.089 Liver 2.85 2.08 0.12 Kidney 2.67 2.48 0.085 Muscle 0.74<0.5 0.008 Fat/kidney 0.82 1.0 <0.005 Heart <0.5 1.09 0.59 Lung 3.701.81 0.020 Brain <0.5 <0.5 Lymph 7.45 16.38 0.12 nodes/trachea lymph1.97 2.74 <0.005 nodes/axillary lymph 7.83 3.8 <0.005 nodes/knee Content(μg/ml) Plasma 0.008 0.035 0.004 Bile 1.96 1.75 0.097

In most of the tissue types studied, the inhibitor was present at from 1to 3 μg/g after 5 to 10 days. 24 h after the last i.p. administration ineach case, the plasma concentrations of the inhibitor were in each caseone to two orders of magnitude below the mean tissue concentrations.From this, high tissue affinity and low plasma protein binding can beconcluded. The concentrations of the naphthylsulfonyl derivative,administered for comparison over 5 days, were 20-30 times lower in thevarious tissues.

The inhibitor shows a noticeable accumulation in lymph nodes. In theindependent experiments, concentrations of 5.3 and 7.5 μg/g,respectively, were measured in tracheal lymph nodes after 5 days ofadministration, and concentrations of 21.6 and 16.4 μg/g, respectively,after 10 days of administration. Since tumor cells often disseminate vialymph tracts, the specific accumulation of the inhibitor in lymphaticvessels is important and advantageous for its use as an anti-metastatictherapeutic agent.

10. UROKINASE inhibitor WX-UK1(N-α(2,4,6-triisopropylphenylsulfonyl)-3-amidino-(L)-phenylalanine-4-ethoxy-carbonylpiperazide-hydrochloride)as Anti-Metastatic Cancer Therapy. Preclinical and Phase I ClinicalData.

In vivo experiments were performed to evaluate the antimetastatic effectof WX-UK1. In WX-UK1-treated animal groups the number of metastaticliver and lung foci was substantially reduced in rat pancreatic andbreast cancer models, respectively. Also, the sizes of subcutaneoustumors were reduced as compared with vehicle-treated controls.

Further the question was adressed, whether WX-UK1 therapy may providebenefits to cancer patients under conditions that may be typicallyoccurring in clinical use such as peri-operatively, at prolonged dosingintervals, or as an add-on therapeutic measure in combination withconventional cytotoxic anti-cancer therapy. The metastatic spread wasused as endpoint in these models.

In detail, the syngeneic transplantable spontaneously metastasizing ratbreast tumor BN472 was used as a model. BN-472 transplantable ratmammary carcinoma used in this study was originally found spontaneouslydeveloped in a retired breeder female Brown Norway rat (Kort et al. J.Nat. Cancer Inst. 1984, 72: 7709-7713). The data presented here focus onthe reduction in the number of metastatic lung foci as endpoint. Theresults are shown in FIGS. 7-9.

FIG. 7 depicts the anti-metastatic efficacy of WX-UK1 in a settingimplying surgical resection of the orthotopically growing tumor at adefined size and various time points of therapy onset relative toresection. After orthotopic implantation of BN472 fragment under themammary fat pad the primary tumors were allowed to grow to a fixedpalpable size (4×4 mm). The treatments groups (n=18) were allocated in away to ensure homogeneous group composition with respect to tumorresidence periods (range 3-14 d) prior group allocation. Primary tumorswere surgically removed 4 days after having reached the fixed size.Onsets of treatments with 0.3 mg/kg s.c. daily were group specificallyeither on the day of reaching the fixed size (PO)mor four (P4), eight(P8), or twelve (P112) days thereafter. Anti-metastatic activity wasobvious with onsets of FIG. 8 displays the results of a BN472 therapystudy aiming at establishing efficacy of WX-UK1 at prolonged dosingintervals. Starting on day 3 after tumor inoculation treatment groups(n=15) received WX-UK1 at weekly doses of 2 mg either by dailyapplication of 0.3 mg/kg (0.3 mg/kg o.d.), by application of 1 mg/kgtwice per week (1 mg/kg t.w.) or by dosing 2 mg/kg once per week (2mg/kg o.w.). The anti-metastatic efficacy previously established withdaily treatments was retained when WX-UK1 was administered only twice oronce per week at the same weekly dose.

FIG. 9 shows additive anti-metastatic benefits of WX-UK1 treatment inthe BN472 cancer model in combination with standard cytotoxic anti-tumortherapies epirubicin or 5-fluorouracie (5-FU). a) Rats (n=18 per group)were treated with vehicle or with 1.5 or 0.75 mg/kg epirubicine i.v.once weekly either alone or in combination with daily S.C. injections of0.3 mg/kg of WX-UK1 or with WX-UK1 monotherapy. Treatment was initiatedon day 3 after tumor inoculation. b) Groups of rats (n=8) were treatedwith 5-FU at 20 or 40 mg/kg i.v. once weekly either as mono-therapy orin combination with WX-UK1 i.p. treatments at 1 mg/kg twice weekly orwith WX-UK1 alone. Indeed, WX-UK1 was efficacious at reducing lung focinumbers when combined with these standard therapeutics.

In a phase I healthy volunteer clinical trial WK-UK1 was well toleratedand demonstrated an advantageous systemic and local safety profile withgood dose proportionality in PK studies.

This phase I study provided the PK and safety data needed to exploreWX-UK1 in cancer patients in a phase I/II trial.

In the first clinical trial with WX-UK1 (WX/50-001) the drug wasinvestigated in a double-blind, randomized, three-way cross-over,placebo controlled phase I study focussing on pharmacokinetics,pharmacodynamics, safety, and tolerability of increasing doses ofWX-UK1.

In this study six ascending i.v. Doses of WX-UK1 in the range of 0.01 to0-3 mg/kg were administered and each dose was given to groups of sixhealthy male volunteers.

Maximum plasma concentrations were in each case seen at the end of the30 min. infusion period (FIG. 10). Thereafter concentrations declinedrapidly and the mean terminal half lives were in the range of 10-12 h.Renal excretion of the patent compounds is less then one percent of thedose.

WX-UK1 was very well tolerated at all dose levels. No changes in vitalsigns. ECG parameters, general safety laboratory parameters, and adverseevent profiles were observed which could be attributed to theadministration of the study drug.

For the coagulation parameters PT, aPTT and TT minor increases wereobserved at doses of 0.05 mg/kg and higher (mean increases of 6-13%after WX-UK1 compared to 0-5% with placebo) and the end of infusion. Allvalues returned to baseline within 15 min. and were regarded notclinically relevant. Bleeding times remained unchanged and there was noindication of a drug-induced hemolysis.

11. Anti-Metastatic Activity f Urokinas Inhibitor WX-UK1 Applied inCombination in Anti-Tumor Therapies with Epirubicin, or 5-FU in the uPA,uPAR and PAI-1 Expressing BN-472 Rat Mammary Carcinoma Model

The effect of various WX-UK1 treatment schedules on tumor growth andtumor spread was studied in vivo using the synergeneic, transplantableand spontaneously metastasizing orthotopic rat mammary tumor model,BN-472 (c.f. Example 10) both as a single agent at various dose levelsas well as in combination with conventional cytotoxic tumor therapeutics(i.e. epirubicin, paclitaxel and S-FU).

To validate the BN-472 rat mammary tumor model for its suitability as atest system for anti-proteolytic treatment modalities the BN-472 tumormodel was characterized by RT-PCR analysis for the expression in tumorsof the target structure i.e. uPA, its cellular receptor uPAR, as well asits physiological uPA-inhibitor PAI-1.

We show the dose dependency of anti-tumor and anti-metastatic effects ofWX-UK1 administrations.

In a series of experiment the additivity regarding anti-tumor andanti-metastatic effects of conventional cytotoxics such as epirubicin,paclitaxel and 5-FU alone or combined with WX-UK1 therapy wasinvestigated.

Materials and Methods

WX-UK1 was dissolved in 5% w/v of D-mannitol to the appropriate dosageand administered once daily or twice weekly either subcutaneously (s.c.)or intraperitonally (i.p.).

Epirubicin (0.75-3 mg/kg body wight), paclitaxel (5-7.5 mg/kg) or 5-FU(20 and 40 mg/kg) was given once weekly by i.v. injection into theexternal jugular vein.

Control rats received the vehicle 5% w/v of D-mannitol once daily s.c.

Tumor Inoculation:

A donor rat bearing a tumor of 20×20 mm was sacrificed, the tumorharvested ant the cortex part was sliced into cubes of approximately 10mm³ and the transplated orthotopically under the mammary fad pads offemale Brown Norway rats.

Treatment was started three or six days (5-FU experiment) after tumorinoculation. Each treatment group consisted of 15 to 21 rats.

Tumor load is plotted as the average±standard error of the mean (SEM).

Tumor, organ weights as well as lung foci counts are presented inBox-Whisker graphs. The box shows the range between 25^(th) and 75^(th)percentiles of values, with a horizontal line at the median value.Whiskers extend to the extreme lower 5^(th) and upper 95^(th)percentiles of values. Dots represent individual data.

P-values represent significance levels of pairwise comparisons oftreatment groups versus vehicle control. For combination therapyexperiments the P-values for the comparison of a cytostatic mono-therapyversus the combination therapy is given.

Results and Discussion

Real-time PCR analysis showed that the BN-472 rat mammary tumorsexpressed among others: uPA, uPAR, PAI-1, MMP-2 and VEGF-A. Theexpression of the plasminogen activator system qualifies BN-472 as asuitable test system for investigating anti-tumor and anti-mestastaticeffects or serine protease-targeting WX-UK1.

Dose-dependent anti-tumor effects were observed upon dailyadministration of WX-UK1 as well as upon once weekly administration ofepirubicin, paclitaxel or 5-FU in BN-472 tumor-bearing rarts regardingtumor size development as well as final tumor weight. cytostaticsepirubicin, paclitaxel and 5-FU resulted in an enhancement of theanti-tumor effects regarding primary tumor size.

Interestingly, in the combination therapies which consist of WX-UK1 andthe cytostatic agents, additional anti-metastatic effects were observed,e.g. less lymph nodes weight or less macroscopic lung foci number.

The results ate shown in FIGS. 11-24.

CONCLUSIONS

Inhibition of tumor growth and mestastatsis formation of BN-472 ratmammary carcinoma by WX-UK1 is in line with the established importanceof these activities for tumor growth and dissemination.

As a single agent, WX-UK1 was more or similarly active as eitherepirubicin, pactitaxel or 5-FU alone at reducing lymphogenic spread asdetermined by lymph-node weight.

The number of macroscopically detectable lung foci was more effectivelyreduced with WX-UK1 than with epirubicin or paclitaxel and was foundequally effective compared with 5-FU. WX-UK1 combined with eitherepirubicin and in particular with 5-FU resulted in statisticallysignificant additive anti-metastatic effects.

The combination of WX-UK1 plus epirubicin, paclitaxel or 5-FU treatmenttended to provide additive benefits regarding the metastatic endpointswith no clue to a synergy of adverse side effects.

The anti-metastatic activity of WX-UK1 was not associated with thymusatrophy as was the case in the epirubicin treated group reflecting itsmode of action other than cytostatic.

Taken together the results obtained in the BN-472 tumor model supportthe combination

1. A method for treating a malignant tumor selected from the groupconsisting of urokinase associated breast cancer and lung foci resultingfrom metastases thereof, and urokinase associated pancreatic cancer andliver foci resulting from metastases thereof, comprising administering acomposition comprisingNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(D,L)-phenylalanine4-ethoxycarbonylpiperazide, the L enantiomer thereof or apharmaceutically suitable salt thereof and a pharmaceutically acceptablecarrier to a patient in need of such treatment.
 2. The method accordingto claim 1, wherein said tumor affects lymphatic tissue.
 3. The methodaccording to claim 2, wherein said lymphatic tissue is lymph nodes. 4.The method according to claim 3, wherein said lymph nodes are selectedfrom the group consisting of axillary lymph nodes and intraperitoneallymph nodes.
 5. The method according to claim 1, further comprisingadministering a cytotoxic substance.
 6. The method according to claim 5,wherein said cytotoxic substance is selected from the group consistingof cisplatin, carboplatin, doxorubicin, epirubicin, 5-fluorouracil and ataxane.
 7. The method according to claim 6, wherein said taxane ispaclitaxel.
 8. The method according to claim 1, wherein said compositionis administered once daily to once weekly.
 9. A pharmaceuticalcomposition comprisingNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(D,L)-phenylalanine4-ethoxycarbonylpiperazide, the L enantiomer thereof or apharmaceutically suitable salt thereof, an additional, pharmacologicallyactive substance, and a pharmaceutically acceptable carrier,wherein saidadditional pharmacologically active substance is selected from the groupconsisting of radio labels or cytotoxic substances.
 10. Thepharmaceutical composition according to claim 9, wherein saidadditional, pharmacologically active substance is a cytotoxic substance.11. The pharmaceutical composition according to claim 10, wherein saidcytotoxic substance is selected from the group consisting of cisplatin,carboplatin, doxorubicin, epirubicin, 5-fluorouracil and a taxane. 12.The pharmaceutical composition according to claim 11, wherein saidtaxane is paclitaxel.
 13. A kit comprising, in separate containers, a)Nα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(D,L)-phenylalanine4-ethoxycarbonylpiperazide, the L enantiomer thereof or apharmaceutically suitable salt thereof, and b) radio labels and/orcytotoxic substances.
 14. A method for the treatment of malignant tumorsselected from the group consisting of urokinase associated breast cancerand lung foci resulting from metastases thereof, and urokinaseassociated pancreatic cancer and liver foci resulting from metastasesthereof comprising a) surgically removing a primary tumor from apatient, and b) administering a composition comprisingNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(D,L)-phenylalanine4-ethoxycarbonylpiperazide, the L enantiomer thereof or apharmaceutically suitable salt thereof and a pharmaceutically acceptablecarrier to said patient.
 15. The method according to claim 14, furthercomprising administering cytotoxic agents and/or radiation therapy tosaid patient.
 16. A method for treating malignant tumors selected fromthe group consisting of urokinase associated breast cancer and lung fociresulting from metastases thereof, and urokinase associated pancreaticcancer and liver foci resulting from metastases thereof, comprisingcontacting a cell with aNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(D,L)-phenylalanine4-ethoxycarbonylpiperazide, the L enantiomer thereof or apharmaceutically suitable salt thereof, wherein said cell is in apatient in need of such treatment.
 17. A method for treating malignanttumors selected from the group consisting of urokinase associated breastcancer and lung foci resulting from metastases thereof, and urokinaseassociated pancreatic cancer and liver foci resulting from metastasesthereof in a patient in need of such treatment, comprising administeringNα(2,4,6-Triisopropylphenylsulfonyl)-3-amidino-(D,L)-phenylalanine4-ethoxycarbonylpiperazide, the L enantiomer thereof or apharmaceutically suitable salt thereof, to a tumor cell in said patient.